Co-optation of Tandem DNA Repeats for the Maintenance of Mesenchymal Identity.

Tandem repeats (TRs) are generated by DNA replication errors and retain a high level of instability, which in principle would make them unsuitable for integration into gene regulatory networks. However, the appearance of DNA sequence motifs recognized by transcription factors may turn TRs into functional cis-regulatory elements, thus favoring their stabilization in genomes. Here, we show that, in human cells, the transcriptional repressor ZEB1, which promotes the maintenance of mesenchymal features largely by suppressing epithelial genes and microRNAs, occupies TRs harboring dozens of copies of its DNA-binding motif within genomic loci relevant for maintenance of epithelial identity. The deletion of one such TR caused quasi-mesenchymal cancer cells to reacquire epithelial features, partially recapitulating the effects of ZEB1 gene deletion. These data demonstrate that the high density of identical motifs in TRs can make them suitable platforms for recruitment of transcriptional repressors, thus promoting their exaptation into pre-existing cis-regulatory networks.

Basis of gene-specific transcription regulation by the Integrator complex.

The Integrator complex attenuates gene expression via the premature termination of RNA polymerase II (RNAP2) at promoter-proximal pausing sites. It is required for stimulus response, cell differentiation, and neurodevelopment, but how gene-specific and adaptive regulation by Integrator is achieved remains unclear. Here, we identify two sites on human Integrator subunits 13/14 that serve as binding hubs for sequence-specific transcription factors (TFs) and other transcription effector complexes. When Integrator is attached to paused RNAP2, these hubs are positioned upstream of the transcription bubble, consistent with simultaneous TF-promoter tethering. The TFs co-localize with Integrator genome-wide, increase Integrator abundance on target genes, and co-regulate responsive transcriptional programs. For instance, sensory cilia formation induced by glucose starvation depends on Integrator-TF contacts. Our data suggest TF-mediated promoter recruitment of Integrator as a widespread mechanism for targeted transcription regulation.

The microRNA-183/96/182 cluster inhibits lung cancer progression and metastasis by inducing an interleukin-2-mediated antitumor CD8+ cytotoxic T-cell response.

One of the mechanisms by which cancer cells acquire hyperinvasive and migratory properties with progressive loss of epithelial markers is the epithelial-to-mesenchymal transition (EMT). We have previously reported that in different cancer types, including nonsmall cell lung cancer (NSCLC), the microRNA-183/96/182 cluster (m96cl) is highly repressed in cells that have undergone EMT. In the present study, we used a novel conditional m96cl mouse to establish that loss of m96cl accelerated the growth of Kras mutant autochthonous lung adenocarcinomas. In contrast, ectopic expression of the m96cl in NSCLC cells results in a robust suppression of migration and invasion in vitro, and tumor growth and metastasis in vivo. Detailed immune profiling of the tumors revealed a significant enrichment of activated CD8+ cytotoxic T lymphocytes (CD8+ CTLs) in m96cl-expressing tumors, and m96cl-mediated suppression of tumor growth and metastasis was CD8+ CTL-dependent. Using coculture assays with naïve immune cells, we show that m96cl expression drives paracrine stimulation of CD8+ CTL proliferation and function. Using tumor microenvironment-associated gene expression profiling, we identified that m96cl elevates the interleukin-2 (IL2) signaling pathway and results in increased IL2-mediated paracrine stimulation of CD8+ CTLs. Furthermore, we identified that the m96cl modulates the expression of IL2 in cancer cells by regulating the expression of transcriptional repressors Foxf2 and Zeb1, and thereby alters the levels of secreted IL2 in the tumor microenvironment. Last, we show that in vivo depletion of IL2 abrogates m96cl-mediated activation of CD8+ CTLs and results in loss of metastatic suppression. Therefore, we have identified a novel mechanistic role of the m96cl in the suppression of lung cancer growth and metastasis by inducing an IL2-mediated systemic CD8+ CTL immune response.

A Zeb1/MtCK1 metabolic axis controls osteoclast activation and skeletal remodeling.

Osteoclasts are bone-resorbing polykaryons responsible for skeletal remodeling during health and disease. Coincident with their differentiation from myeloid precursors, osteoclasts undergo extensive transcriptional and metabolic reprogramming in order to acquire the cellular machinery necessary to demineralize bone and digest its interwoven extracellular matrix. While attempting to identify new regulatory molecules critical to bone resorption, we discovered that murine and human osteoclast differentiation is accompanied by the expression of Zeb1, a zinc-finger transcriptional repressor whose role in normal development is most frequently linked to the control of epithelial-mesenchymal programs. However, following targeting, we find that Zeb1 serves as an unexpected regulator of osteoclast energy metabolism. In vivo, Zeb1-null osteoclasts assume a hyperactivated state, markedly decreasing bone density due to excessive resorptive activity. Mechanistically, Zeb1 acts in a rheostat-like fashion to modulate murine and human osteoclast activity by transcriptionally repressing an ATP-buffering enzyme, mitochondrial creatine kinase 1 (MtCK1), thereby controlling the phosphocreatine energy shuttle and mitochondrial respiration. Together, these studies identify a novel Zeb1/MtCK1 axis that exerts metabolic control over bone resorption in vitro and in vivo.

RNA binding protein ZCCHC24 promotes tumorigenicity in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) lacks the expression of hormone and HER2 receptors and is highly malignant with no effective therapeutic targets. In TNBC, the cancer stem-like cell (CSC) population is considered to be the main cause of resistance to treatment. Thus, the therapeutic targeting of this population could substantially improve patient survival. Here, we identify the RNA-binding protein ZCCHC24 as enriched in the mesenchymal-like TNBC population. ZCCHC24 promotes the expression of a set of genes related to tumorigenicity and treatment resistance by directly binding to the cis-element "UGUWHWWA" in their mRNAs, thereby stabilizing them. One of the ZCCHC24 targets, ZEB1, is a transcription factor that promotes the expression of cancer stemness genes and reciprocally induces ZCCHC24 expression. ZCCHC24 knockdown by siRNAs shows a therapeutic effect and reduces the mesenchymal-like cell population in TNBC patient-derived xenografts. ZCCHC24 knockdown also has additive effects with the BET inhibitor JQ1 in suppressing tumor growth in TNBC patient-derived xenografts.

The adaptive antioxidant response during fasting-induced muscle atrophy is oppositely regulated by ZEB1 and ZEB2.

Reactive oxygen species (ROS) serve important homeostatic functions but must be constantly neutralized by an adaptive antioxidant response to prevent supraphysiological levels of ROS from causing oxidative damage to cellular components. Here, we report that the cellular plasticity transcription factors ZEB1 and ZEB2 modulate in opposing directions the adaptive antioxidant response to fasting in skeletal muscle. Using transgenic mice in which Zeb1 or Zeb2 were specifically deleted in skeletal myofibers, we show that in fasted mice, the deletion of Zeb1, but not Zeb2, increased ROS production and that the adaptive antioxidant response to fasting essentially requires ZEB1 and is inhibited by ZEB2. ZEB1 expression increased in fasted muscles and protected them from atrophy; conversely, ZEB2 expression in muscles decreased during fasting and exacerbated muscle atrophy. In fasted muscles, ZEB1 reduces mitochondrial damage and increases mitochondrial respiratory activity; meanwhile, ZEB2 did the opposite. Treatment of fasting mice with Zeb1-deficient myofibers with the antioxidant triterpenoid 1[2-cyano-3,12-dioxool-eana-1,9(11)-dien-28-oyl] trifluoro-ethylamide (CDDO-TFEA) completely reversed their altered phenotype to that observed in fasted control mice. These results set ZEB factors as potential therapeutic targets to modulate the adaptive antioxidant response in physiopathological conditions and diseases caused by redox imbalance.

YAP/TAZ-mediated regulation of laminin 332 is enabled by ß4 integrin repression of ZEB1 to promote ferroptosis resistance.

We are interested in the contribution of integrins and the extracellular matrix to epithelial differentiation in carcinomas. This study was motivated by our finding that the Hippo effectors YAP and TAZ can sustain the expression of laminin 332 (LM332), the predominant ECM ligand for the integrin ß4, in breast carcinoma cells with epithelial differentiation. More specifically, we observed that YAP and TAZ regulate the transcription of the LAMC2 subunit of LM332. Given that the ß4-LM332 axis is associated with epithelial differentiation and YAP/TAZ have been implicated in carcinoma de-differentiation, we sought to resolve this paradox. Here, we observed that the ß4 integrin sustains the expression of miR-200s that target the transcription factor ZEB1 and that ZEB1 has a pivotal role in determining the nature of YAP/TAZ-mediated transcription. In the presence of ß4, ZEB1 expression is repressed enabling YAP/TAZ/TEAD-mediated transcription of LAMC2. The absence of ß4, however, induces ZEB1, and ZEB1 binds to the LAMC2 promoter to inhibit LAMC2 transcription. YAP/TAZ-mediated regulation of LAMC2 has important functional consequences because we provide evidence that LM332 enables carcinoma cells to resist ferroptosis in concert with the ß4 integrin.

Dynamic EMT: a multi-tool for tumor progression.

The process of epithelial-mesenchymal transition (EMT) is fundamental for embryonic morphogenesis. Cells undergoing it lose epithelial characteristics and integrity, acquire mesenchymal features, and become motile. In cancer, this program is hijacked to confer essential changes in morphology and motility that fuel invasion. In addition, EMT is increasingly understood to orchestrate a large variety of complementary cancer features, such as tumor cell stemness, tumorigenicity, resistance to therapy and adaptation to changes in the microenvironment. In this review, we summarize recent findings related to these various classical and non-classical functions, and introduce EMT as a true tumorigenic multi-tool, involved in many aspects of cancer. We suggest that therapeutic targeting of the EMT process will-if acknowledging these complexities-be a possibility to concurrently interfere with tumor progression on many levels.

Roles of ZEB1 and ZEB2 in E-cadherin expression and cell aggressiveness in head and neck cancer.

Zinc finger E-box binding homeobox 1 (ZEB1) has been identified as a key factor in cancer cell differentiation and metastasis, and has been well studied in the field of cancer cell biology. ZEB2 has a highly similar conformation to ZEB1, but its role in head and neck squamous cell carcinoma (HNSCC) cells is not fully understood. Here, we separately overexpressed ZEB1 and ZEB2 in C57BL/6 mouse oral cancer (MOC) cells and investigated their cellular characteristics, including E-cadherin levels, motile properties, chemoresistance, and metastatic ability in immunocompetent mice. Both ZEB1 and ZEB2 overexpression reduced epithelial traits and converted cells to an aggressive phenotype. Surprisingly, ZEB1 overexpression increased the endogenous level of ZEB2 in MOC cells, and vice versa. The molecular mechanisms underlying these findings remain unclear. However, the in vitro anchorage-independent growth of MOC cells overexpressing ZEB2 was considerably greater than that of MOC cells overexpressing ZEB1. These findings suggest that ZEB2, like ZEB1, has the ability to induce the differentiation of cancer cells into those with highly aggressive traits.

GTSE1-driven ZEB1 stabilization promotes pulmonary fibrosis through the epithelial-to-mesenchymal transition.

G2 and S phase-expressed protein 1 (GTSE1) has been implicated in the development of pulmonary fibrosis (PF); however, its biological function, molecular mechanism, and potential clinical implications remain unknown. Here, we explored the genomic data of patients with idiopathic PF (IPF) and found that GTSE1 expression is elevated in their lung tissues, but rarely expressed in normal lung tissues. Thus, we explored the biological role and downstream events of GTSE1 using IPF patient tissues and PF mouse models. The comprehensive bioinformatics analyses suggested that the increase of GTSE1 in IPF is linked to the enhanced gene signature for the epithelial-to-mesenchymal transition (EMT), leading us to investigate the potential interaction between GTSE1 and EMT transcription factors. GTSE1 preferentially binds to the less stable form of zinc-finger E-box-binding homeobox 1 (ZEB1), the unphosphorylated form at Ser585, inhibiting ZEB1 degradation. Consistently, the ZEB1 protein level in IPF patient and PF mouse tissues correlates with the GTSE1 protein level and the amount of collagen accumulation, representing fibrosis severity. Collectively, our findings highlight the GTSE1-ZEB1 axis as a novel driver of the pathological EMT characteristic during PF development and progression, supporting further investigation into GTSE1-targeting approaches for PF treatment.

Cytolethal Distending Toxin Modulates Cell Differentiation and Elicits Epithelial to Mesenchymal Transition.

BACKGROUND: The bacterial genotoxin, cytolethal distending toxin (CDT), causes DNA damage in host cells, a risk factor for carcinogenesis. Previous studies have shown that CDT induces phenotypes reminiscent of epithelial to mesenchymal transition (EMT), a process involved in cancer initiation and progression. METHODS: We investigated different steps of EMT in response to Helicobacter hepaticus CDT and its active CdtB subunit using in vivo and in vitro models. RESULTS: Most of the steps of the EMT process were induced by CDT/CdtB and observed throughout the study in murine and epithelial cell culture models. CdtB induced cell-cell junction disassembly, causing individualization of cells and acquisition of a spindle-like morphology. The key transcriptional regulators of EMT (SNAIL and ZEB1) and some EMT markers were upregulated at both RNA and protein levels in response to CDT/CdtB. CdtB increased the expression and proteolytic activity of matrix metalloproteinases, as well as cell migration. A range of these results were confirmed in Helicobacter hepaticus-infected and xenograft murine models. In addition, colibactin, a genotoxic metabolite produced by Escherichia coli, induced EMT-like effects in cell culture. CONCLUSIONS: Overall, these data show that infection with genotoxin-producing bacteria elicits EMT process activation, supporting their role in tumorigenesis.

Trophoblast cell surface antigen-2 phosphorylation triggered by binding of galectin-3 drives metastasis through down-regulation of E-cadherin.

The expression of trophoblast cell surface antigen-2 (Trop-2) is enhanced in many tumor tissues and is correlated with increased malignancy and poor survival of patients with cancer. Previously, we demonstrated that the Ser-322 residue of Trop-2 is phosphorylated by protein kinase Cα (PKCα) and PKCδ. Here, we demonstrate that phosphomimetic Trop-2 expressing cells have markedly decreased E-cadherin mRNA and protein levels. Consistently, mRNA and protein of the E-cadherin-repressing transcription factors zinc finger E-Box binding homeobox 1 (ZEB1) were elevated, suggesting transcriptional regulation of E-cadherin expression. The binding of galectin-3 to Trop-2 enhanced the phosphorylation and subsequent cleavage of Trop-2, followed by intracellular signaling by the resultant C-terminal fragment. Binding of ß-catenin/transcription factor 4 (TCF4) along with the C-terminal fragment of Trop-2 to the ZEB1 promoter upregulated ZEB1 expression. Of note, siRNA-mediated knockdown of ß-catenin and TCF4 increased the expression of E-cadherin through ZEB1 downregulation. Knockdown of Trop-2 in MCF-7 cells and DU145 cells resulted in downregulation of ZEB1 and subsequent upregulation of E-cadherin. Furthermore, wild-type and phosphomimetic Trop-2 but not phosphorylation-blocked Trop-2 were detected in the liver and/or lung of some nude mice bearing primary tumors inoculated intraperitoneally or subcutaneously with wild-type or mutated Trop-2 expressing cells, suggesting that Trop-2 phosphorylation, plays an important role in tumor cell mobility in vivo, too. Together with our previous finding of Trop-2 dependent regulation of claudin-7, we suggest that the Trop-2-mediated cascade involves concurrent derangement of both tight and adherence junctions, which may drive metastasis of epithelial tumor cells.

The aryl sulfonamide indisulam inhibits gastric cancer cell migration by promoting the ubiquitination and degradation of the transcription factor ZEB1.

Gastric cancer is one of the cancers with high morbidity and mortality worldwide. The aryl sulfonamide indisulam inhibits the proliferation of several types of cancer cells through its function as a molecular glue to promote the ubiquitination and degradation of RNA-binding motif protein 39 (RBM39). However, it is unknown whether and how indisulam regulates the migration of cancer cells. In this work, using label-free quantitative proteomics, we discover that indisulam significantly attenuates N-cadherin, a marker for epithelial to mesenchymal transition and migration of cancer cells. Our bioinformatics analysis and biochemical experiments reveal that indisulam promotes the interaction between the zinc finger E-box-binding homeobox 1 (ZEB1), a transcription factor of N-cadherin, and DCAF15, a substrate receptor of CRL4 E3 ubiquitin ligase, and enhances ZEB1 ubiquitination and proteasomal degradation. In addition, our cell line-based experiments demonstrate that indisulam inhibits the migration of gastric cancer cells in a ZEB1-dependent manner. Analyses of patient samples and datasets in public databases reveal that tumor tissues from patients with gastric cancer express high ZEB1 mRNA and this high expression reduces patient survival rate. Finally, we show that treatment of gastric tumor samples with indisulam significantly reduces ZEB1 protein levels. Therefore, this work discloses a new mechanism by which indisulam inhibits the migration of gastric cancer cells, indicating that indisulam exhibits different biological functions through distinct signaling molecules.

NF-κB/RelA signaling in secretoglobin progenitors mediates plasticity and MMP-induced barrier disruption in house dust mite-induced allergic asthma.

The mechanisms how aeroallergens induce sensitization are incompletely understood. The house dust mite (HDM) Dermatophagoides pteronyssius (Der p) is a ubiquitous aeroallergen that represents a major cause of allergic rhinitis and asthma. Herein, we tested whether HDM-induced aeroallergen exposure sensitivity is caused by the innate-immune response in small airway epithelial cells. HDM exposure is a rapid activator of NF-κB/RelA in the Secretoglobin (Scgb1a1+) lineage associated with upregulation of NF-κB/RelA-dependent markers of epithelial plasticity. To determine the effect of epithelial NF-κB signaling, NF-κB was depleted in a tamoxifen (TMX)-inducible Scgb1a1-CreERTM mouse within a CL57B/L6 background. Corn oil or TMX-treated/RelA-depleted [RelA knockdown (KD)] mice were repetitively exposed to airway HDM challenges to induce airway hyperresponsiveness (AHR). Strikingly, we observed that HDM induces hallmarks of epithelial plasticity through upregulation of the mesenchymal core factors SNAI1 and ZEB1 and production of metalloproteinase (MMP)9 that are RelA-dependent. Downstream, HDM-induced mucous metaplasia, Th2 polarization, allergen sensitivity, and airway hyperreactivity were all reduced in the RelA-depleted mice. Mechanistically, HDM-induced functional and structural barrier disruption was dependent on RelA signaling and associated with active MMP secretion into the bronchoalveolar lavage fluid. To establish the role of MMP2/9 in barrier disruption, we observe that a small-molecule MMP inhibitor (SB-3CT) blocked HDM-induced barrier disruption and activation of plasticity in naïve wild-type (WT) mice. Loss of functional barrier was associated with MMP disruption of zona occludens (ZO)-1 containing adherens junctions. Overall, this data indicates that host innate signaling in the Scgb1a1+ progenitors is directly linked to epithelial plasticity, MMP9 secretion, and enhanced barrier permeability that allows allergen penetration, sensitization producing allergic asthma (AA) in vivo. We propose that maintenance of epithelial integrity may reduce allergic sensitization and AA.NEW & NOTEWORTHY Allergic asthma from house dust mite (HDM) allergy causes substantial morbidity. This study examines the dynamic changes in small airway epithelial cells in a mouse model of HDM exposure. Our findings indicate that NF-κB/RelA signaling mediates matrix metalloproteinase production, disrupting the epithelial barrier resulting in allergic sensitization. Our findings bring new insight into mechanisms for epithelial cell-state change in the allergen response, creating a potential therapeutic pathway for maintaining barrier function in asthma.

PrLZ regulates EMT and invasion in prostate cancer via the TGF-ß1/p-smad2/miR-200 family/ZEB1 axis.

BACKGROUND: Prostate leucine zipper (PrLZ) is a prostate-specific protein, and our previous study demonstrated that PrLZ enhances the malignant progression of prostate cancer (Pca). However, the roles of PrLZ in epithelial to mesenchymal transition (EMT) remain unknown. METHODS: Quantitative real-time PCR (qRT-PCR), immunohistochemical (IHC) staining, hematoxylin-eosin (HE) staining, and western blotting were used to analyze the expression of protein and genes level in human PCa cell lines. Invasion assay was used to examine the effect of PrLZ, miR-200a, miR-200b, miR-200c, miR-141, miR-429, miR-205, and ZEB1 on PCa cell line invasion in vitro. Prostate cancer metastasis animal model was designed to assess the effect of PrLZ on PCa cell line invasion in vivo. RESULTS: We proved that high PrLZ expression initiates EMT, which was shown by the downregulation of E-cadherin and upregulation of vimentin in PC-3/PrLZ and ARCaP-E/PrLZ cells. Mechanistic analysis revealed that PrLZ regulates EMT by activating TGF-ß1/p-smad2 signaling and further inhibiting the expression of miR-200 family members, which negatively regulates ZEB1 expression and causes EMT in Pca. Moreover, using two of orthotopic mouse model and tail vein injection of human prostate cancer cells mouse model, we observed that PC-3/PrLZ cells led to the development of distant organ metastases in vivo. CONCLUSIONS: Our results show the mechanism by which PrLZ regulates EMT and metastasis and suggest that PrLZ may be a potential therapeutic target for Pca metastasis.

Genome-wide cooperation of EMT transcription factor ZEB1 with YAP and AP-1 in breast cancer.

Invasion, metastasis and therapy resistance are the major cause of cancer-associated deaths, and the EMT-inducing transcription factor ZEB1 is a crucial stimulator of these processes. While work on ZEB1 has mainly focused on its role as a transcriptional repressor, it can also act as a transcriptional activator. To further understand these two modes of action, we performed a genome-wide ZEB1 binding study in triple-negative breast cancer cells. We identified ZEB1 as a novel interactor of the AP-1 factors FOSL1 and JUN and show that, together with the Hippo pathway effector YAP, they form a transactivation complex, predominantly activating tumour-promoting genes, thereby synergising with its function as a repressor of epithelial genes. High expression of ZEB1, YAP, FOSL1 and JUN marks the aggressive claudin-low subtype of breast cancer, indicating the translational relevance of our findings. Thus, our results link critical tumour-promoting transcription factors: ZEB1, AP-1 and Hippo pathway factors. Disturbing their molecular interaction may provide a promising treatment option for aggressive cancer types.

Extracellular matrix marker LAMC2 targets ZEB1 to promote TNBC malignancy via up-regulating CD44/STAT3 signaling pathway.

BACKGROUND: Triple negative breast cancer (TNBC) is a heterogeneous and aggressive disease characterized by a high risk of mortality and poor prognosis. It has been reported that Laminin γ2 (LAMC2) is highly expressed in a variety of tumors, and its high expression is correlated with cancer development and progression. However, the function and mechanism by which LAMC2 influences TNBC remain unclear. METHODS: Kaplan-Meier survival analysis and Immunohistochemical (IHC) staining were used to examine the expression level of LAMC2 in TNBC. Subsequently, cell viability assay, wound healing and transwell assay were performed to detect the function of LAMC2 in cell proliferation and migration. A xenograft mouse model was used to assess tumorigenic function of LAMC2 in vivo. Luciferase reporter assay and western blot were performed to unravel the underlying mechanism. RESULTS: In this study, we found that higher expression of LAMC2 significantly correlated with poor survival in the TNBC cohort. Functional characterization showed that LAMC2 promoted cell proliferation and migration capacity of TNBC cell lines via up-regulating CD44. Moreover, LAMC2 exerted oncogenic roles in TNBC through modulating the expression of epithelial-mesenchymal transition (EMT) markers. Luciferase reporter assay verified that LAMC2 targeted ZEB1 to promote its transcription. Interestingly, LAMC2 regulated cell migration in TNBC via STAT3 signaling pathway. CONCLUSION: LAMC2 targeted ZEB1 via activating CD44/STAT3 signaling pathway to promote TNBC proliferation and migration, suggesting that LAMC2 could be a potential therapeutic target in TNBC patients.

Effect of infection by Mycoplasma arginini and Mycoplasma salivarium on the oncogenic properties of lung cancer cell line A549.

Most mycoplasma species are the extracellular parasites affecting different cellular processes including proliferation, cell cycle, protein synthesis, DNA repair and others. Mycoplasma infection was shown to contribute to the pathology of various diseases, including cancer. Upon infection, mycoplasmas typically activate the tumor-associated NF-kB pathway, which is associated with EMT, the main mechanism of metastasis. In this study, we found that two different mycoplasma strains, M. arginini and M. salivarium, promoted the initiation of EMT and simultaneous suppression of the p53 tumor suppressor in A549 lung cancer cells. This led to an increase of cancer cell motility, resistance to the antitumor drug etoposide concomitantly with decreased autophagy. These data indicate that mycoplasmas are able to increase the tumorigenic potential of cancer host cells.

GLUT3-mediated cigarette smoke-induced epithelial-mesenchymal transition in chronic obstructive pulmonary disease through the NF-kB/ZEB1 pathway.

BACKGROUND: Airway remodelling plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Epithelial-mesenchymal transition (EMT) is a significant process during the occurrence of airway remodelling. Increasing evidence suggests that glucose transporter 3 (GLUT3) is involved in the epithelial mesenchymal transition (EMT) process of various diseases. However, the role of GLUT3 in EMT in the airway epithelial cells of COPD patients remains unclear. METHODS: We detected the levels of GLUT3 in the peripheral lung tissue of COPD patients and cigarette smoke (CS)-exposed mice. Two Gene Expression Omnibus GEO datasets were utilised to analyse GLUT3 gene expression profiles in COPD. Western blot and immunofluorescence were used to detect GLUT3 expression. In addition, we used the AAV9-GLUT3 inhibitor to reduce GLUT3 expression in the mice model. Masson's staining and lung function measurement were used detect the collagen deposition and penh in the mice. A cell study was performed to confirm the regulatory effect of GLUT3. Inhibition of GLUT3 expression with siRNA, Western blot, and immunofluorescence were used to detect the expression of E-cadherin, N-cadherin, vimentin, p65, and ZEB1. RESULTS: Based on the GEO data set analysis, GLUT3 expression in COPD patients was higher than in non-smokers. Moreover, GLUT3 was highly expressed in COPD patients, CS exposed mice, and BEAS-2B cells treated with CS extract (CSE). Further research revealed that down-regulation of GLUT3 significantly alleviated airway remodelling in vivo and in vitro. Lung function measurement showed that GLUT3 reduction reduced airway resistance in experimental COPD mice. Mechanistically, our study showed that reduction of GLUT3 inhibited CSE-induced EMT by down-regulating the NF-κB/ZEB1 pathway. CONCLUSION: We demonstrate that CS enhances the expression of GLUT3 in COPD and further confirm that GLUT3 may regulate airway remodelling in COPD through the NF-κB/ZEB1 pathway; these findings have potential value in the diagnosis and treatment of COPD. The down-regulation of GLUT3 significantly alleviated airway remodelling and reduced airway resistance in vivo. Our observations uncover a key role of GLUT3 in modulating airway remodelling and shed light on the development of GLUT3-targeted therapeutics for COPD.

PD-L1 knockdown suppresses vasculogenic mimicry of non-small cell lung cancer by modulating ZEB1-triggered EMT.

BACKGROUND: PD-L1 overexpression is commonly observed in various malignancies and is strongly correlated with poor prognoses for cancer patients. Moreover, PD-L1 has been shown to play a significant role in promoting angiogenesis and epithelial-mesenchymal transition (EMT) processes across different cancer types. METHODS: The relationship between PD-L1 and vasculogenic mimicry as well as epithelial-mesenchymal transition (EMT) was explored by bioinformatics approach and immunohistochemistry. The functions of PD-L1 in regulating the expression of ZEB1 and the EMT process were assessed by Western blotting and q-PCR assays. The impact of PD-L1 on the migratory and proliferative capabilities of A549 and H1299 cells was evaluated through wound healing, cell invasion, and CCK8 assays following siRNA-mediated PD-L1 knockdown. Tube formation assay was utilized to evaluate the presence of VM structures. RESULTS: In this study, increased PD-L1 expression was observed in A549 and H1299 cells compared to normal lung epithelial cells. Immunohistochemical analysis revealed a higher prevalence of VM structures in the PD-L1-positive group compared to the PD-L1-negative group. Additionally, high PD-L1 expression was also found to be significantly associated with advanced TNM stage and increased metastasis. Following PD-L1 knockdown, NSCLC cells exhibited a notable reduction in their ability to form tube-like structures. Moreover, the levels of key EMT and VM-related markers, including N-cadherin, MMP9, VE-cadherin, and VEGFA, were significantly decreased, while E-cadherin expression was upregulated. In addition, the migration and proliferation capacities of both cell lines were significantly inhibited after PD-L1 or ZEB1 knockdown. CONCLUSIONS: Knockdown PD-L1 can inhibit ZEB1-mediated EMT, thereby hindering the formation of VM in NSCLC.